Giardia lamblia is the most common disease-causing parasite in the United States responsible for an estimated 3 million cases a year. The surface proteins of Giardia change in the test tube as well as in infections in animals and man and is likely important for the survival of the parasite in humans and animals.The mechanisms Girardia lamblia employs to switch its surface antigens are unknown and have been difficult to study. The developement and use of transfection techniques in Giardia have allowed the expression of foreign or native proteins, inhibition of proteins or the placement of proteins of specific interest into the selected spots in the genome. A method was developed that allowed studies to understand how only one Giardia surface antigen(VSP) could be expressed while another very similar surface antigen is not. Group 1(WB) and Group 3(GS) Giardia isolates express unique variant-specific surface proteins (VSP) including WB1267 and VSPH7, respectively. Although vspH7 could be expressed on the surface of WB using constitutive promoters, vspH7 expression was not appropriately transcribed when transfected into WB using its own 5' and 3' flanking sequences either as an episome or when integrated into the WB genome. To determine whether vspH7 was correctly transcribed in the homologous GS isolate, an influenza hemagglutinin epitope (HA) was introduced into vspH7 (vspH7-HA) and along with its 5' and 3' flanking sequences transfected and subsequently integrated into the genome. VspH7 was appropriately expressed and the transcription initiation site was identical to native vspH7. By PFGE analysis both vspH7-HA and vspH7 localized to chromosome V and sequence analysis of the upstream 3 kb of both vsps showed they were virtually the same at 99% identical but could be differentiated from each other by unique sequences further upstream. VSPH7-HA expression could be distinguished from expression of VSPH7 by the presence of the HA epitope in addition to expression of a H7 specific epitope on the same trophozoite. VSPH7-HA and VSPH7 were expressed independently and analysis of cultures expressing mostly one or the other vsp by Southern blots after restriction with several enzymes revealed no change in the band size of either culture. These experiments suggest that both vspH7 and vspH7-HA are present in homologous sites on chromosome V and transcription from each is controlled independently in the absence of gene movement. These data indicate epigenetic mechanisms control surface antigenic variation.